MSACL 2025 Abstract

Decoding the Glycan Signature: Unraveling N-Glycosylation Alterations in Glycogen Storage Disease Ia and Ib

Ruiqi Xiao (1), Hector F. B. R. Loponte (2,3,4), Jing Zheng (2), Maaike H. Oosterveer(1,5,6), Terry G.J. Derks(7), Peter L. Horvatovich (2), M. Rebecca Heiner-Fokkema (8), Barbara M. Bakker(1), Justina C. Wolters(1), Guinevere S. M. Lageveen-Kammeijer(2)
(1) Laboratory of Pediatrics, Department of Pediatrics, University Medical Center Groningen; Groningen, The Netherlands, (2) Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen; Groningen, The Netherlands, (3) Carlos Chagas Filho Biophysics’ Institute, Federal University of Rio de Janeiro; Rio de Janeiro, Brazil, (4) Paulo de Góes Microbiology Institute, Federal University of Rio de Janeiro; Rio de Janeiro, Brazil, (5) Laboratory Medicine, University of Groningen, University Medical Center Groningen; Groningen, The Netherlands, (6) Therapy Accelerator for Rare Diseases, Radboud University Medical Center, Nijmegen, The Netherlands, (7) Beatrix Children’s Hospital, Department of Pediatrics, University of Groningen, University Medical Center Groningen; Groningen, The Netherlands, (8) Laboratory of Metabolic Diseases, Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen; Groningen, The Netherlands

Guinevere Lageveen-Kammeijer, PhD (Presenter) University of Groningen

Presenter Bio: Dr. Guinevere Lageveen-Kammeijer is an Assistant Professor in the Analytical Biochemistry group at the University of Groningen, within the Groningen Research Institute of Pharmacy. She holds a BSc in Biotechnology - Forensic Sciences from the University of Applied Sciences van Hall Larenstein, Leeuwarden, and an MSc in Analytical Chemistry from VU University, Amsterdam. Her research interests were ignited during her MSc internship, where she focused on separation techniques coupled with mass spectrometry.

Guinevere earned her PhD in Clinical Glycomics from the Leiden University Medical Center in 2019 under the supervision of Prof. Manfred Wuhrer. Her thesis developed small-scale sample preparation workflows using capillary electrophoresis (CE) and mass spectrometry (MS/MS) to analyze glycans, glycopeptides, and glycoproteins, with applications in biomarker discovery and biopharmaceutical characterization. She continued her research as a post-doctoral researcher at the same institution before expanding her expertise with a visit to Northeastern University, Boston, in 2017, where she focused on protein charge and proteoform heterogeneity.

In 2022, Guinevere began her tenure-track assistant professorship at the University of Groningen, where she works on advancing glyco(proteo)mic techniques, particularly in single-cell glycomic analysis. Her research includes expanding the mass spectrometry-based glycosylation assay for prostate-specific antigen (PSA), a key biomarker for prostate cancer, and exploring the in-depth analysis of glycans and glycoproteins for biomarker discovery in other diseases and biopharmaceutical characterization.

Guinevere’s contributions have been recognized through funding such as the Investigator Sponsored Research grant from Astellas (2019) and the prestigious NWO VENI grant (2023). She is actively involved in the scientific community, serving on the Scientific Omics Committee for MSACL. Guinevere is passionate about promoting the importance of glycosylation in biomarker research, aiming to bridge the gap between researchers and clinical professionals to improve biomarker translation to the clinic.

Relevant Financial Disclosures (within past 24 months, reported on Mar 18, 2025) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Glycogen storage disease (GSD) types Ia and Ib are rare metabolic disorders caused by pathogenic variants in G6PC and SLC37A4, leading to deficiencies in glucose-6-phosphatase (G6PC) and glucose-6-phosphate transporter (G6PT), respectively. These defects disrupt glucose homeostasis, which may in turn impact glycosylation—a fundamental cellular process that relies on glycosyl donors synthesized in glucose-dependent pathways. Beyond its dependence on glucose availability, glycosylation plays a crucial role in protein folding, stability, and cell signaling, all of which are essential for proper cellular function. Disruptions in glycosylation have been implicated in immune dysfunction, organ damage, and disease progression in other metabolic disorders, suggesting that glycomic alterations could contribute to the pathology of GSD as well. However, the impact of dysfunctional glucose metabolism on N-glycosylation across the proteome in GSD I patients remains largely unexplored. This study addresses this gap by systematically analyzing plasma N-glycomic alterations in GSD Ia and Ib, aiming to reveal new insights into disease pathology and potential therapeutic targets.

METHODS:
To evaluate whether N-glycomic changes occur, we conducted glycomic analysis on serum/plasma samples of 12 GSD Ia and 5 GSD Ib patients, including for some patients various timepoints, resulting in a total of 18 GSD Ia and 8 GSD Ib samples. Healthy control samples (n=21) were age- and gender-matched. All samples were collected retrospectively. An in-solution N-glycan release from the glycoproteins in the samples was performed using the enzyme PNGase F, followed by sialic acid derivatization and neutralization (ethyl esterification and amidation). After purifying the derivatized N-glycans by cotton HILIC-SPE, the samples were labeled with a permanent cationic label (Girard’s reagent P). Using high-sensitivity capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) the N-glycome for each sample was identified, characterized and quantified, providing insights into glycomic alterations associated with GSD Ia and Ib. The data was curated by setting the minimum isotopic fitting to 90%, curve fitting to 90%, minimal signal-to-noise of 3 and a maximum ppm error deviation of 10.

RESULTS/DISCUSSION:
Our preliminary analysis provided valuable insights into the glycan biosynthesis in relation to GSD. In total, 249 distinct N-glycans were identified and quantified, of which 174 were detected in all three groups. Notably, 21 and 20 N-glycan compositions were unique to GSD Ia and GSD Ib patients, respectively. While 28 N-glycans were shared between both patient groups but absent in controls. This extensive glycomic diversity highlights the profound alterations in glycan biosynthesis, likely driven by metabolic dysfunctions in GSD. Early findings indicate that GSD Ia patients exhibit an upregulation of complex, multi-antennary N-glycans compared to healthy controls, a trend that appears less pronounced in GSD Ib patients. Additionally, 42 N-glycans were found to be significantly different between GSD Ia and GSD Ib groups, suggesting that each subtype exhibits a distinct glycomic signature. These observations emphasize the need for further investigation to determine whether specific glycan features correlate with altered biosynthetic pathways and impaired glucose metabolism in GSD.

CONCLUSION:
This study presents the first in-depth characterization of the serum or plasma N-glycomic landscape in GSD Ia and Ib patients. Our findings reveal significant glycosylation alterations, which will offer new perspectives on metabolic and biosynthetic disruptions in GSD. Future research will focus on elucidating the mechanistic links between glycosylation changes as well as its correlation with previously obtained proteomics data. This will help us to further refine our understanding of glycan modifications in GSD and by identifying distinct glycomic signatures, this research will contribute to the development of targeted therapeutic strategies and will pave the way for improving clinical outcomes for GSD patients.